Mix gently by pipetting up and down or flicking the tube 4-5 times. Do not vortex. Place the mixture on ice for 30 minutes. Do not mix.

Heat shock at 42°C for 30 seconds. Do not mix.

Transfer tubes on ice for 2 minutes.

Add 950 µl of room temperature SOC media to tubes.

Place the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.

Warm selection plates to 37°C.

Spread 100 µl of the cells onto the plates with appropriate antibiotics. Use amp plates for positive control sample.

Incubate plates overnight at 37°C.

SHuffle Express Competent E. coli (C3028)

Thaw a tube of SHuffle Competent E. coli cells on ice for 10 minutes.

Add 1–5 µl containing 1 pg–100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4–5 times to mix cells and DNA. Do not vortex.

Place the mixture on ice for 30 minutes. Do not mix.

Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.

Place on ice for 5 minutes. Do not mix.

Pipette 950 µl of room temperature SOC into the mixture.

Place at 30°C for 60 minutes. Shake vigorously (250 rpm) or rotate.

Warm selection plates to 30°C.

Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.

Spread 50–100 µl of each dilution onto a selection plate and incubate overnight at 30°C. Alternatively, incubate at 25°C for 48 hours.

Qiagen MiniPrep

For overnight cell cultures 1~5mL of E. Coli in LB medium:

Transfer cell culture into centrifuge tubes

Centrifuge for 5 min. at 4,000 rpm

Discard the supernatant

Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.

Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Do not vortex. Continue inverting the tube until the solution becomes viscous + slightly clear blue. Do not allow lysis reaction to proceed for more than 5 min.

Add 350 µl Buffer N3 and mix immediately + thoroughly by 4-6x inverting the tube. The solution should become cloudy.

BIO-RAD Protein Gel

Get DTT (in the biology section of the desiccator in the fridge--which is on the second shelf from bottom right side).

Mix 54mg of DTT in 950µL of loading dye.

Determine amount of protein and buffer in each lane according to following table: Note that the protein amount CANNOT exceed 8µg per lane for 10 lane gel, and 6µg per lane for 15 lane gel. Get 1.5mL microtubes as many as needed.

Heat the tubes for 5-10 min, and get the ladder (marked "P" at the top in the Alaska fridge, in the box "Joshi Gel ladders/dyes") out to thaw.

While the tubes are being heated, prepare the gel:

Take the green cap off the wells and clean the wells with water. Repeat washing about three times. Take the tape off bottom.

Assemble the cassette with two gels or (one gel + a gel-wall). Note that the wells should face inwards.

Put the cassette into the gel box and pour NEW Tris-gly-SDS buffer into the cassette past. The buffer level should be past the wells. Check for any leaking. If there is a leak, reassemble the cassette and try again.

Set up the voltage machine to the desired voltage and time. For a fast run, 190V with 35 minutes could work. For slow and cleaner result, 90V with 50min could work.

Now get the heated samples, and the ladder. Add 10µL of ladder for 15 lane gels, 15µL of ladder for 10 lane gels. Add 15µL of the sample for 15 lane gels, 30µL for of the 10 lane gels.

Fill the box to the 2-gel mark (or 4-gel mark if running more than 3 gels) or more with OLD Tris-gly-SDS buffer. Put the cap on and hit the run button. Check for bubbles rising.

Coomassie Staining

Retrieve the gel; be careful not to rip, and if sticky apply water. Put it in an empty box.

Check the staining solution (50% methanol, 40% water, 10% acetic acid, 0.25mg per 100mL) in the fume-hood in the back corridor.

If the caps have been open, or the solution is out, make a new one (500mL) by:

50% of total volume is methanol (is in the flammable cabinet in the chemistry room). 40% of total volume is water. Use plastic graduated cylinder for these.

10% of total volume is acetic acid (is in the acid cabinet). BE CAUTIOUS WHEN WALKING CORNERS, and switch gloves after touching acid. Use GLASS graduated cylinder for this.

measure out and add the Coomassie to appropriate amount. It is located in the "Bahamas"

NOTE: methanol waste must go to the special waste bin. If the waste bin is full, call the number.